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Published on: July 22, 2020
Molecular techniques for studying gene expression in carcinogenesis
1Department of Radiation Oncology, Leo W Jenkins Cancer Center, The Brody School of Medicine, East Carolina University, Greenville, NC 27858. ahmedf@mail.ecu.edu
Abstract:
Many genes and signaling pathways controlling cell proliferation, death, differentiation, and genomic integrity are involved in cancer development. Various methods are available for detection and quantification of messenger RNA. Older methods such as Northern blots, nuclease protection, plaque hybridization, and slot blots suffer from being inherently serial, measure a single mRNA at a time, or being difficult to automate. New techniques for analysis of gene expression include: (a) comprehensive open systems such as serial analysis of gene expression (SAGE), differential display (DD) analysis, RNA arbitrarily primer (RAP)-PCR, restriction endonucleolytic analysis of differentially expressed sequences (READS), amplified restriction fragment-length polymorphism (AFLP), total gene expression analysis (TOGA), and use of internal standard competitive template primers (CTs) in a quantitative multiplex RT-PCR method [StaRT-(PCR)], and (b) focused closed systems such as: high density cDNA filter hybridization (HDFCA) analysis, suppression subtractive hybridization (SSH), differential screening (DS), several forms of high-density cDNA arrays, or oligonucleotide chips, and tissue microarrays. Sometimes, a combination of these systems is used to enhance the sensitivity and specificity of the assays. While closed systems are excellent for the initial screening of large number of sequences, the value of the information generated is generally limited to an often arbitrarily chosen known sequence. On the other hand, only the open system platform has the potential to evaluate the expression patterns of tens of thousands of genes that have not yet been cloned or partially sequenced in a quantitative manner. A cost analysis of the most commonly used expression technologies is provided. A method for purifying tumors from surrounding stroma and normal tissue employing laser microdissection, and subsequent RNA isolation/amplification from few cells employing sensitive kits are also discussed.
Insights
This study compares traditional and novel gene expression analysis techniques for cancer research. Open systems offer broader gene discovery potential than closed systems, aiding in understanding cancer development.
Area of Science:
- Molecular Biology
- Cancer Research
- Genomics
Background:
- Cancer development involves complex gene and signaling pathway regulation.
- Accurate messenger RNA (mRNA) detection and quantification are crucial for cancer research.
- Traditional mRNA analysis methods have limitations in automation and throughput.
Purpose of the Study:
- To review and compare various gene expression analysis technologies for cancer research.
- To highlight the advantages of open systems for comprehensive gene discovery.
- To discuss methods for tumor sample preparation and RNA isolation.
Main Methods:
- Comparison of traditional methods (Northern blots, etc.) with novel open systems (SAGE, DD, etc.) and closed systems (cDNA arrays, chips, etc.).
- Discussion of combined system approaches for enhanced sensitivity and specificity.
- Inclusion of cost analysis for commonly used expression technologies.
Main Results:
- Open systems provide quantitative analysis of numerous unknown genes, essential for discovery.
- Closed systems are effective for screening known sequences but offer limited discovery scope.
- Laser microdissection and sensitive RNA isolation kits enable analysis from limited tumor samples.
Conclusions:
- Open systems are superior for unbiased, large-scale gene expression profiling in cancer research.
- The choice of technology depends on the research question, balancing discovery with targeted analysis.
- Advanced sample preparation techniques improve the feasibility of gene expression studies on small tumor samples.
